Peptides for the treatment of oxidative stress related disorders

ABSTRACT

Isolated DJ-1 related peptides are disclosed and pharmaceutical compositions comprising same for treating oxidative stress-related disorder.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/127,494 filed on May 4, 2011, which is a National Phase of PCT PatentApplication No. PCT/IL2010/000760 having International filing date ofSep. 16, 2010, which claims the benefit of priority of U.S. ProvisionalPatent Application No. 61/272,367 filed on Sep. 17, 2009. The contentsof the above applications are all incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to peptideagents for the treatment of oxidative stress related disorders.

Free radicals are extremely reactive chemical species that causesignificant destruction in biological systems. Indiscriminate reactionof free radicals with biological molecules can lead to the destructionof cells and cellular components (e.g. mitochondria), thereby affectingphysiological processes by causing cells to lose their structure and/orfunction.

In biological systems, free radicals are generally referred to as‘reactive oxygen species’ (ROS). ROS are derived from endogenous sourcesvia the metabolism of oxygen containing species, and from exogenoussources such as toxins and atmospheric pollutants.

Attack of ROS on biological molecules is referred to as ‘oxidativestress’. Oxidative stress has been implicated as a causative factor in anumber of degenerative diseases.

Parkinson's disease (PD) is a multifactorial disease caused by bothgenetic and environmental factors. Although most patients suffering fromPD have a sporadic disease, several genetic causes have been identifiedin recent years. An increasing number of genes that cause inheritedforms of PD have provided the opportunity for new insights into themechanisms at the basis of the disease. These genes includealpha-synuclein, parkin, PINK1, dardarin (LRRK2), and DJ-1.

Current concepts of the pathogenesis of PD center on the formation ofROS and the onset of oxidative stress leading to oxidative damage tosubstantia nigra pars compacta. Extensive postmortem studies haveprovided evidence to support the involvement of oxidative stress in thepathogenesis of PD; in particular, these include alterations in brainiron content, impaired mitochondrial function, alterations in theantioxidant protective systems (most notably superoxide dismutase [SOD]and reduced glutathione [GSH]), and evidence of oxidative damage tolipids, proteins, and DNA. Iron can induce oxidative stress, andintranigral injections have been shown to induce a model of progressiveparkinsonism.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of thecentral nervous system (CNS), characterized by various symptoms ofneurological dysfunction. MS and its animal model, experimentalautoimmune encephalomyelitis (EAE), are believed to result fromautoimmune mediated activated immune cells such as T- and B-lymphocytesas well as macrophages and microglia. Pathologically, MS ischaracterized by perivenous infiltration of lymphocytes and macrophagesinto the CNS parenchyma, resulting in demyelinative lesions termedplaques. These plaques, which are the hallmark of MS, are associatedwith oligodendrocytes death, axonal damage and neuronal loss. The viewthat MS can be considered an inflammatory neurodegenerative disease issupported by studies demonstrating neuronal and axonal injury in regionsremote from acute plaques, as well as imaging studies that demonstratedchanges in normal appearing white and grey matter.

The etiology of MS has not yet been fully elucidated, and it has beenattributed to both genetic and environmental causes. Accumulating dataindicate that oxidative stress plays a major role in the pathogenesis ofMS. Reactive oxygen species (ROS), leading to oxidative stress,generated in excess primarily by activated microglia, have beenimplicated as mediators of demyelination and axonal damage in both MSand EAE.

The neurotransmitter glutamate is one of the sources of oxidative stressin the MS primarily through activation of its ionotropic receptors.Oligodendrocytes, the myelin-producing cell of the CNS, are also highlyvulnerable to glutamate excitotoxicity, mainly via the AMPA/kainatereceptors. ROS causes damage to cardinal cellular components such aslipids, proteins and nucleic acids, resulting in cell death. Weakenedcellular antioxidant defense systems in the CNS of MS patients resultingin increased vulnerability to ROS effects may increase CNS damage.

Amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease,is a progressive, fatal neurological disease affecting as many as 30,000Americans with 5,000 new cases occurring in the United States each year.The disorder belongs to a class of disorders known as motor neurondiseases. ALS occurs when specific nerve cells in the brain and spinalcord that control voluntary movement gradually degenerate. Familialamyotrophic lateral sclerosis (FALS) is a form of ALS distinguished fromthe more common sporadic variant only by its familial background.

There is substantial evidence to support the hypothesis that oxidativestress is a contributor to motor neuron death. For example, it has beendiscovered that mutation of the anti-oxidant enzyme, superoxidedismutase 1 (SOD1), causes disease in a significant minority of cases.

DJ-1 is a small 189 amino acid protein that is ubiquitously expressedand highly conserved throughout diverse species. Accumulating datarevealed its involvement in various cellular processes, especially inoxidative stress. DJ-1 is known to have several isoforms withisoelectric points between 5.5 and 7, with dominance of alkalineisoforms in normal conditions. Upon ROS exposure there is accumulationof more acidic isoforms of DJ-1, mediated through oxidation of cysteineresidues [Bandopadhyay R, et al., Brain 127: 420-430, 2004; Canet-Avileset al., Proc Natl Acad Sci USA 101: 9103-9108, 2004].

DJ-1 is widely distributed and is highly expressed in the brain, and isnot confined to a single functional system or anatomical location. DJ-1is expressed in neurons of different neurotransmitter phenotypes and inall glial cell types, such as astrocytes, microglia andoligodendrocytes. Recently DJ-1 mutations were discovered and associatedwith familial Parkinson's disease (PD) [Bonifati et al., 2003, Science299: 256-9, 2003]. A post-mortem study of brain samples from sporadic PDbrains versus control found that acidic isoforms of DJ-1 are moreabundant in PD brains [Bandopadhyay R, et al., Brain 127: 420-430,2004]. DJ-1 immunoreactivity was detected in other neurodegenerativediseases including multi-system atrophy, Alzheimer's disease,progressive supranuclear palsy, fronto-temporal dementia withparkinsonism linked to chromosome 17, and Pick's disease [BandopadhyayR, et al., Brain 127: 420-430, 2004; Neumann N. et al., Acta Neuropathol(Berl) 107: 489-496, 2004; Rizzu P. et al., Ann Neurol 55: 113-118,2004].

WO2007/119237 discusses an analysis of DJ-1 levels and activity fordiagnosing oxidative stress related disorders.

U.S. Pat. Appl. No. 20060153807 and U.S. Pat. Appl. No. 20060171935discusses vector mediated gene regulation of, e.g., DJ-1-associatedagents for the treatment of neurodegenerative diseases such asParkinson's.

WO2008/111063 discusses peptide agents capable of up-regulatingDJ-1-dependent VMAT2 transcription for the treatment ofneurodegenerative diseases.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided an isolated peptide being no longer than 25 aminoacids comprising a sequence as set forth in SEQ ID NO: 3, wherein theisolated peptide increases viability of a cell under oxidative stressconditions.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising an isolatedpeptide, or peptide mimetic thereof, no longer than 25 amino acidscomprising at least 2 consecutive amino acids from the amino acidsequence as set forth in SEQ ID NO: 1, and a pharmaceutically acceptablecarrier, wherein the isolated peptide increases viability of a cellunder oxidative stress conditions.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising an isolatedpeptide, or peptide mimetic thereof, no longer than 25 amino acidscomprising at least 2 consecutive amino acids from the amino acidsequence as set forth in SEQ ID NO: 2, wherein the isolated peptideincreases viability of a cell under oxidative stress conditions.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating an oxidative stress-relateddisorder in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of thepharmaceutical composition of the present invention, thereby treatingthe oxidative stress-related disorder.

According to an aspect of some embodiments of the present inventionthere is provided an isolated peptide, or peptide mimetic thereof, nolonger than 25 amino acids comprising at least 2 consecutive amino acidsfrom the amino acid sequence as set forth in SEQ ID NO: 1, and apharmaceutically acceptable carrier, wherein the isolated peptideincreases viability of a cell under oxidative stress conditions.

According to an aspect of some embodiments of the present inventionthere is provided an isolated peptide, or peptide mimetic thereof, nolonger than 25 amino acids comprising at least 2 consecutive amino acidsfrom the amino acid sequence as set forth in SEQ ID NO: 2, wherein theisolated peptide increases viability of a cell under oxidative stressconditions.

According to some embodiments of the invention, the isolated peptidecomprises a sequence as set forth in SEQ ID NO:3.

According to some embodiments of the invention, the isolated peptidecomprises a sequence as set forth in SEQ ID NO:14.

According to some embodiments of the invention, the isolated peptide isas set forth in SEQ ID NO: 24.

According to some embodiments of the invention, the isolated peptidecomprises at least 5 consecutive amino acids from the amino acidsequence as set forth in SEQ ID NO: 1.

According to some embodiments of the invention, the isolated peptidecomprises at least 5 consecutive amino acids from the amino acidsequence as set forth in SEQ ID NO: 2.

According to some embodiments of the invention, the isolated peptidecomprises no more than 15 consecutive amino acids from the amino acidsequence as set forth in SEQ ID NO: 1.

According to some embodiments of the invention, the isolated peptidecomprises comprising no more than 15 consecutive amino acids from theamino acid sequence as set forth in SEQ ID NO: 2.

According to some embodiments of the invention, the isolated peptide isno longer than 20 amino acids.

According to some embodiments of the invention, the isolated peptidecomprises a sequence as set forth in SEQ ID NO: 38.

According to some embodiments of the invention, the isolated peptidecomprises a sequence as set forth in SEQ ID NO: 39.

According to some embodiments of the invention, the isolated peptidecomprises a sequence as set forth in SEQ ID NO: 10.

According to some embodiments of the invention, the isolated peptidecomprises a sequence as set forth in SEQ ID NO: 11.

According to some embodiments of the invention, the isolated peptidecomprises a sequence as set forth in SEQ ID NO: 1 or 3.

According to some embodiments of the invention, the isolated peptidecomprises a sequence as set forth in SEQ ID NO: 40.

According to some embodiments of the invention, the isolated peptidecomprises a sequence as set forth in SEQ ID NO: 13.

According to some embodiments of the invention, the isolated peptidecomprises a sequence as set forth in SEQ ID NO: 41.

According to some embodiments of the invention, the isolated peptidecomprises a sequence as set forth in SEQ ID NO: 42.

According to some embodiments of the invention, the isolated peptidecomprises a sequence as set forth in SEQ ID NO: 2.

According to some embodiments of the invention, at least one of theamino acids is a naturally occurring amino acid.

According to some embodiments of the invention, at least one of theamino acids is a synthetic amino acid.

According to some embodiments of the invention, the isolated peptide isattached to a cell penetrating agent.

According to some embodiments of the invention, the attached iscovalently attached.

According to some embodiments of the invention, the cell penetratingagent is a peptide agent.

According to some embodiments of the invention, the peptide cellpenetrating agent comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 21-23.

According to some embodiments of the invention, the isolated peptide isas set forth in SEQ ID NOs: 24-36.

According to some embodiments of the invention, the cell comprises aneuronal cell.

According to some embodiments of the invention, the ROS conditions areselected from the group consisting of 6-hydroxydopamine toxicity,hydrogen peroxide toxicity, UV radiation and dopamine toxicity.

According to some embodiments of the invention, the oxidativestress-related disorder is a neurodegenerative disease.

According to some embodiments of the invention, the neurodegenerativedisease is selected from the group consisting of Parkinson's disease,Multiple Sclerosis, ALS, multi-system atrophy, Alzheimer's disease,stroke, progressive supranuclear palsy, fronto-temporal dementia withparkinsonism linked to chromosome 17 and Pick's disease.

According to some embodiments of the invention, the isolated peptide isfor use in treating an oxidative stress-related disorder.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying images. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-F are bar graphs illustrating that DJ-1-related peptide TAT 2a(SEQ ID NO: 24) is protective in vitro against oxidative and toxicinsults. Increased cell viability (A), decreased mitochondrial damage(B) and increased metabolic activity (C) were found with DJ-1-relatedpeptide treatment as compared to saline alone, after exposure tooxidative stress, induced by exposure to H₂O₂. Cell proliferation wasmeasured by quantifying [³H]thymidine incorporation. There was noincreased cell proliferation with exposure to the peptides (D).Protection against 6-hydroxydopamine and dopamine toxicity are shown inFIGS. 1E and F, respectively. The presented experiments were done onhuman neuroblastoma SH—SY5Y cells, and repeated at least 3 times.*p<0.05, as compared to control cells treated with vehicle and exposedto the same toxic insult.

FIGS. 2A-B are bar graphs illustrating that DJ-1-related peptides areprotective in vitro against 6-hydroxydopamine toxicity. Increasedmetabolic activities were found when human neuroblastoma cells SH—SY5Ywere pretreated with DJ-1-related peptides as compared to saline beforeexposure to 6-hydroxydopamine, Protection against 6-hydroxydopaminetoxicity by derivatives of DJ-1 related peptide #2 are shown in FIG. 2Aand protection against 6-hydroxydopamine toxicity by derivatives of DJ-1related peptide #5 are shown in FIG. 2B. Mutated peptides failed toprotect against 6-hydroxydopamine toxicity as shown in FIGS. 2A-B. Thepresented experiments were done on human neuroblastoma SH—SY5Y cells,and repeated at least 3 times. *p<0.05, as compared to control cellsexposed to the same toxic insult.

FIG. 3 illustrates that DJ-1-related peptides are protective in vitroagainst 6-hydroxydopamine toxicity. Increased viability was found whenhuman neuroblastoma cells SH—SY5Y were pretreated with DJ-1-relatedpeptide II (SEQ ID NO: 8) as compared to saline before exposure to6-hydroxydopamine, *p<0.05, as compared to control cells exposed to thesame toxic insult.

FIG. 4A is a bar graph illustrating that DJ-1-related peptides areprotective against serum deprivation. Increased viability was found whenhuman neuroblastoma cells SH—SY5Y were pretreated with DJ-1-relatedpeptide I (SEQ ID NO: 10), II (SEQ ID NO: 8) and short 2a (SEQ ID NO: 9)as compared to saline during serum deprivation, *p<0.05.

FIG. 4B is a bar graph illustrating that control DJ-1 related peptidesare not protective against oxidative and toxin-induced toxicity. Cellswere exposed to increasing doses of 6-hydroxydopamine (0-25 uM) or tohydrogen peroxide (H₂O₂, 0-50 uM) with or without pretreatment withcontrol peptides X and Y (sequences that were not prospected to induceneuroprotection). The presented experiments were done on humanneuroblastoma SH—SY5Y cells, and repeated 3 times. No significantdifferences were observed compared to control cells treated with vehicleand exposed to the same toxic dose.

FIGS. 5A-B are bar graphs illustrating protection against H₂O₂ and6-hydroxydopamine toxicity by TAT 2a (SEQ ID NO: 24) peptide in primarycultures. Similar protection against increasing concentrations ofhydrogen peroxide and 6-hydroxydopamine was demonstrated in primarymixed neuronal and glial cultures obtained from cortex of postnatal wildtype C57/bl mice. Each experiment was repeated for 3 times. *p<0.05, ascompared to control cells treated with vehicle and exposed to the sametoxic insult.

FIG. 6 is a bar graph illustrating DJ-1-related peptides I (SEQ ID NO:23) and II (SEQ ID NO: 8) are protective against oxidative insult inneuronally differentiated murine neural stem cells. Increased viabilitywas found when these cells were pretreated with DJ-1-related peptide Ior II as compared to saline before exposure to hydrogen peroxide (H₂O₂),*p<0.05, as compared to control cells exposed to the same toxic insult.

FIGS. 7A-B are photographs illustrating immunohistochemical staining foralpha-synuclein in naïve neuroblastoma cells (FIG. 7A) and cells stablytransfected with A53T mutant alpha-synuclein (FIG. 7B). Alpha-synucleinstains red by Alexa-568 conjugated secondary antibodies. Nuclei werecounterstained by DAPI (blue). The experiment was repeated 3 times intriplicates.

FIG. 8 is a bar graph illustrating graphic quantification ofalpha-synuclein and a photograph of a representative Western blot foralpha-synuclein in mock transfected neuroblastoma cells and cells stablytransfected with wild type (WT) alpha-synuclein or A53T mutantalpha-synuclein. The experiment was repeated 3 times in triplicates.

FIG. 9 is a bar graph illustrating protection against 6-hydroxydopaminetoxicity by derivatives of peptide #2 in cells overexpressing mutantalpha-synuclein. Significantly increased metabolic activity wasdemonstrated in neuroblastoma cells overexpressing A53T alpha-synucleinexposed to 6-hydroxydopamine, when treated with derivatives ofDJ-1-related peptide #2. Each experiment was repeated for 3 times.*p<0.05, as compared to control cells treated with vehicle and exposedto the same toxic insult.

FIG. 10 is a bar graph illustrating protection against hydrogen peroxidetoxicity by peptide TAT 2a (SEQ ID NO: 24) in cells overexpressingmutant alpha-synuclein. Significantly increased viability wasdemonstrated in neuroblastoma cells overexpressing A53T alpha-synucleinexposed to hydrogen peroxide, when treated with DJ-1-related peptide TAT2a (SEQ ID NO: 24). *p<0.05, as compared to control cells treated withvehicle and exposed to the same toxic insult.

FIG. 11 is a bar graph illustrating that DJ-1-related peptides areprotective in vitro against dopamine toxicity. Cells were exposed toincreasing doses of dopamine (0-50 uM) with or without pretreatment withderivatives of DJ-1 related peptide #2. The presented experiments weredone on human neuroblastoma SH—SY5Y cells, and repeated 3 times.*p<0.05, as compared to control cells treated with vehicle and exposedto the same dopamine dose.

FIGS. 12A-B are bar graphs illustrating that DJ-1-related peptides areprotective against hydrogen peroxide and SIN-I toxicity in NSC-34 cells.Increased viability was found when NSC-34 cells were pretreated withDJ-1-related peptides TAT 2a (SEQ ID NO: 24) or TAT 2b (SEQ ID NO: 25)as compared to saline before exposure to hydrogen peroxide (FIG. 12A) orSIN-I (FIG. 12B) toxicity, *p<0.05, **p<0.01 as compared to controlcells exposed to the same toxic insult.

FIG. 13 is a bar graph illustrating that derivatives of DJ-1-relatedpeptide #2 are protective in a cellular model of ALS. Increasedmetabolic activities were found when NSC-34 cells were pretreated withderivatives of DJ-1-related peptide #2 as compared to saline beforeexposure to SIN-I toxicity, Mutated peptide #2 failed to protect againstSIN-I toxicity as shown. The presented experiment was repeated 3 times.*p<0.05, as compared to control cells exposed to the same toxic insult.

FIG. 14 is a bar graph illustrating that DJ-1-related peptides areprotective in a cellular model against UV radiation. Increased metabolicactivities were found when HaCaT cells were pretreated with DJ-1-relatedpeptide II (SEQ ID NO: 8) or TAT 2a (SEQ ID NO:24) as compared to salinebefore exposure to UV radiation. The presented experiment was repeated 3times. *p<0.05, as compared to control.

FIGS. 15A-B are bar graphs illustrating that intrastriatal TAT 2apeptide injection protects against in vivo 6-hydroxydopaminehemiparkinsonian mice model. 2 μl of 100 μM peptide TAT 2a (SEQ ID NO:24) dissolved in saline or saline alone were injected stereotacticallyinto the right striatum one hour before 6-hydroxydopamine lesioning.Significantly reduced amphetamine-induced rotations at 2 and 4-weeksafter the lesioning was found in the peptide treated mice as compared tocontrols,*p<0.01 (FIG. 15A). Moreover, a smaller fraction of thelesioned mice rotated in response to amphetamine injection (FIG. 15B).The experiment was repeated twice.

FIGS. 16A-B are bar graphs illustrating that intravenous (IV) peptideTAT 2a (SEQ ID NO: 24) injection protects against in vivo6-hydroxydopamine hemiparkinsonian mice model. 50 μg peptide TAT 2a (SEQID NO: 24) or saline alone were injected IV 4 hours before6-hydroxydopamine lesioning. Significantly reduced amphetamine-inducedrotations were demonstrated in the IV peptide treated mice as comparedto controls, *p<0.01 (FIG. 16A). A smaller fraction of the lesioned micerotated in response to amphetamine injection (FIG. 16B).

FIG. 17 is a bar graph illustrating that subcutaneous injection ofpeptide (SEQ ID NO: 25) protects against MPTP Parkinson's model.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

Many neurodegenerative disorders are believed to be associated withoxidative stress such as Parkinson's disease and stroke. Accumulatingdata has revealed that DJ-1, a small 189 amino acid protein is involvedvarious cellular processes, especially in oxidative stress and its usefor the treatment of neurodegenerative diseases has been proposed.

The present disclosure, in some embodiments thereof, relates to DJ-1related peptides that are useful for treating oxidative stress-relateddisorders. Two minimal core sequences on the DJ-1 protein have beenidentified in connection with the present disclosure which comprisetherapeutic activity. A number of peptides have been prepared whichincluded one or more of such core sequences and neuroprotectingproperties have been demonstrated.

Specifically, it has been found that DJ-1 related peptides comprising atleast one of the two core sequences are able to protect against toxicinsults including 6-hydroxydopamine, dopamine and H₂O₂ in in vitro cellsystems (FIGS. 1A-F, 2A-B, 3, 11, 12A), as well as serum depravation(FIG. 4A), whilst other DJ-1 related peptides which do not compriseeither of the identified core sequences, comprise no suchneuroprotective properties (FIG. 4B). The experiments were repeated inprimary cultures (FIGS. 5A-B) and differentiated neural stem cells (FIG.6) with the same results.

Another aspect of the present disclosure relates to an in vitro cellmodel of Parkinson's disorder which overexpresses mutant A53Talpha-synuclein in neuroblastoma cells. DJ-1 related peptides ofembodiments of the present disclosure were demonstrated to be protectiveagainst 6-hydroxydopamine toxicity (FIG. 9) and H₂O₂ toxicity (FIG. 10)in the in vitro cell model.

Yet another aspect of the present disclosure relates to a cellular modelfor ALS and UV irradiation, in which DJ-1 related peptides ofembodiments of the present disclosure were demonstrated to be protectivein these assays as well (FIGS. 13 and 14).

Finally, DJ-1 related peptides of embodiments of the present disclosureare tested in an in vivo model of Parkinson's disease. Certain DJ-1related peptides showed therapeutic activity following both local (FIGS.15A-B) and systemic administration (FIGS. 16A-B).

Altogether, in accordance with the present disclosure, it has been foundthat peptides derived from DJ-1 (“DJ-1 related peptides) can be used forthe treatment of oxidative stress related disorders. The phrase“oxidative stress conditions” as used herein, refers to conditions thatelevate the level of reactive oxidative species (ROS) beyond the normallevel. As mentioned this may result from a lack of antioxidants or froman over abundance free radicals. Exemplary ROS conditions include, butare not limited to 6-hydroxydopamine toxicity, hydrogen peroxidetoxicity, UV radiation and dopamine toxicity.

In certain embodiments, certain aspects of the disclosure, as describedin more full detail herein, relate to short DJ-1 related peptides nolonger than 20 amino acids in length. More particularly, according toone aspect of the present disclosure, there is provided an isolated DJ-1related peptide, or peptide mimetic thereof, no longer than 25 aminoacids comprising at least 2 consecutive amino acids from the amino acidsequence as set forth in SEQ ID NO: 1, wherein the isolated peptideincreases viability of a cell under oxidative stress conditions.

KGAEEMETVIPVDVMRRAGI—(SEQ ID NO: 1; also referred to herein as #peptide2)

According to embodiments of this aspect of the present disclosure, thepeptide comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 consecutive amino acids from the amino acidsequence as set forth in SEQ ID NO: 1.

-   -   KGAEEMETVIPVDVMRRAGI—(SEQ ID NO: 1)

According to one embodiment of this aspect of the present disclosure,the peptide comprises no more than 10 consecutive amino acids of SEQ IDNO: 1.

According to other embodiments of this aspect of the present disclosure,the peptide comprises no more than 15 consecutive amino acids of SEQ IDNO: 1.

Exemplary sequences comprised in the peptide of this aspect of thepresent disclosure include SEQ ID NOs: 1-12, SEQ ID NO: 38 or SEQ ID NO:39.

According to another aspect of the present disclosure, there is providedan isolated peptide, or peptide mimetic thereof, no longer than 25 aminoacids comprising at least 2 consecutive amino acids from the amino acidsequence as set forth in SEQ ID NO: 2, wherein the isolated peptideincreases viability of a cell under oxidative stress conditions.

EGPYDVVVLPGGNLGAQNLS—(SEQ ID NO: 2; also referred to herein as peptide#5)

According to embodiments of this aspect of the present disclosure, thepeptide comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 consecutive amino acids from the amino acidsequence as set forth in SEQ ID NO: 2.

According to one embodiment of this aspect of the present disclosure thepeptide comprises no more than 10 consecutive amino acids of SEQ ID NO:2.

According to other embodiments of this aspect of the present disclosurethe peptide comprises no more than 15 consecutive amino acids of SEQ IDNO: 2.

Exemplary sequences comprised in the peptide of this aspect of thepresent disclosure include SEQ ID NOs: 2, 13-20, 40-42.

The term “peptide” as used herein refers to a polymer of natural orsynthetic amino acids, encompassing native peptides (either degradationproducts, synthetically synthesized polypeptides or recombinantpolypeptides) and peptidomimetics (typically, synthetically synthesizedpeptides), as well as peptoids and semipeptoids which are polypeptideanalogs, which may have, for example, modifications rendering thepeptides even more stable while in a body or more capable of penetratinginto cells.

Such modifications include, but are not limited to N terminusmodification, C terminus modification, polypeptide bond modification,including, but not limited to, CH2-NH, CH2-S, CH2-S═O, O═C—NH, CH2-O,CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications, and residuemodification. Methods for preparing peptidomimetic compounds are wellknown in the art and are specified, for example, in Quantitative DrugDesign, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press(1992), which is incorporated by reference as if fully set forth herein.Further details in this respect are provided hereinunder.

Polypeptide bonds (—CO—NH—) within the polypeptide may be substituted,for example, by N-methylated bonds (—N(CH3)-CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2-), α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH2-NH—), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),polypeptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the polypeptidechain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine(Nol), ring-methylated derivatives of Phe, halogenated derivatives ofPhe or o-methyl-Tyr.

In addition to the above, the polypeptides of the present invention mayalso include one or more modified amino acids or one or more non-aminoacid monomers (e.g. fatty acids, complex carbohydrates etc).

As used herein in the specification and in the claims section below theterm “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids (stereoisomers).

Tables 1 and 2 below list naturally occurring amino acids (Table 1) andnon-conventional or modified amino acids (Table 2) which can be usedwith the present invention.

TABLE 1 Three-Letter One-letter Amino Acid Abbreviation Symbol alanineAla A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic Acid Glu E glycine Gly G Histidine His Hisoleucine Iie I leucine Leu L Lysine Lys K Methionine Met Mphenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr Ttryptophan Trp W tyrosine Tyr Y Valine Val V Any amino acid as above XaaX

TABLE 2 Non-conventional amino acid Code Non-conventional amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgincarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α ethylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcyclopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cyclododeclglycine Ncdod D-α-methylalnine Dnmala N-cyclooctylglycineNcoct D-α-methylarginine Dnmarg N-cyclopropylglycine NcproD-α-methylasparagine Dnmasn N-cycloundecylglycine NcundD-α-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-α-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylleucine Dnmleu N-(3-indolylyethyl) glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvaD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α thylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomo phenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α ethylhistidine Mhis L-α-methylhomophenylalanine Mhphe L-αthylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-α-methylleucineMleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine mser L-α-methylthreonine Mthr L-α ethylvaline MtrpL-α-methyltyrosine Mtyr L-α-methylleucine MvalL-N-methylhomophenylalanine Nmhphe

nbhm N-(N-(2,2-diphenylethyl) N-(N-(3,3-diphenylpropyl)carbamylmethyl-glycine Nnbhm carbamylmethyl(1)glycine Nnbhe1-carboxy-1-(2,2-diphenyl Nmbc ethylamino)cyclopropane

indicates data missing or illegible when filed

The amino acids of the peptides of the present invention may besubstituted either conservatively or non-conservatively.

The term “conservative substitution” as used herein, refers to thereplacement of an amino acid present in the native sequence in thepeptide with a naturally or non-naturally occurring amino or apeptidomimetics having similar steric properties. Where the side-chainof the native amino acid to be replaced is either polar or hydrophobic,the conservative substitution should be with a naturally occurring aminoacid, a non-naturally occurring amino acid or with a peptidomimeticmoiety which is also polar or hydrophobic (in addition to having thesame steric properties as the side-chain of the replaced amino acid).

As naturally occurring amino acids are typically grouped according totheir properties, conservative substitutions by naturally occurringamino acids can be easily determined bearing in mind the fact that inaccordance with the invention replacement of charged amino acids bysterically similar non-charged amino acids are considered asconservative substitutions.

For producing conservative substitutions by non-naturally occurringamino acids it is also possible to use amino acid analogs (syntheticamino acids) well known in the art. A peptidomimetic of the naturallyoccurring amino acid is well documented in the literature known to theskilled practitioner.

When affecting conservative substitutions the substituting amino acidshould have the same or a similar functional group in the side chain asthe original amino acid.

The phrase “non-conservative substitutions” as used herein refers toreplacement of the amino acid as present in the parent sequence byanother naturally or non-naturally occurring amino acid, havingdifferent electrochemical and/or steric properties. Thus, the side chainof the substituting amino acid can be significantly larger (or smaller)than the side chain of the native amino acid being substituted and/orcan have functional groups with significantly different electronicproperties than the amino acid being substituted. Examples ofnon-conservative substitutions of this type include the substitution ofphenylalanine or cycohexylmethyl glycine for alanine, isoleucine forglycine, or —NH—CH[(—CH₂)₅—COOH]—CO— for aspartic acid. Thosenon-conservative substitutions which fall under the scope of the presentinvention are those which still constitute a peptide havinganti-bacterial properties.

As mentioned, the N and C termini of the peptides of the presentinvention may be protected by function groups. Suitable functionalgroups are described in Green and Wuts, “Protecting Groups in OrganicSynthesis”, John Wiley and Sons, Chapters 5 and 7, 1991, the teachingsof which are incorporated herein by reference. Preferred protectinggroups are those that facilitate transport of the compound attachedthereto into a cell, for example, by reducing the hydrophilicity andincreasing the lipophilicity of the compounds.

These moieties can be cleaved in vivo, either by hydrolysis orenzymatically, inside the cell. Hydroxyl protecting groups includeesters, carbonates and carbamate protecting groups. Amine protectinggroups include alkoxy and aryloxy carbonyl groups, as described abovefor N-terminal protecting groups. Carboxylic acid protecting groupsinclude aliphatic, benzylic and aryl esters, as described above forC-terminal protecting groups. In one embodiment, the carboxylic acidgroup in the side chain of one or more glutamic acid or aspartic acidresidue in a peptide of the present invention is protected, preferablywith a methyl, ethyl, benzyl or substituted benzyl ester.

Examples of N-terminal protecting groups include acyl groups (—CO—R1)and alkoxy carbonyl or aryloxy carbonyl groups (—CO—O—R1), wherein R1 isan aliphatic, substituted aliphatic, benzyl, substituted benzyl,aromatic or a substituted aromatic group. Specific examples of acylgroups include acetyl, (ethyl)-CO—, n-propyl-CO—, iso-propyl-CO—,n-butyl-CO—, sec-butyl-CO—, t-butyl-CO—, hexyl, lauroyl, palmitoyl,myristoyl, stearyl, oleoyl phenyl-CO—, substituted phenyl-CO—,benzyl-CO— and (substituted benzyl)-CO—. Examples of alkoxy carbonyl andaryloxy carbonyl groups include CH3-O—CO—, (ethyl)-O—CO—,n-propyl-O—CO—, iso-propyl-O—CO—, n-butyl-O—CO—, sec-butyl-O—CO—,t-butyl-O—CO—, phenyl-O—CO—, substituted phenyl-O—CO— and benzyl-O—CO—,(substituted benzyl)-O—CO—. Adamantan, naphtalen, myristoleyl, tuluen,biphenyl, cinnamoyl, nitrobenzoy, toluoyl, furoyl, benzoyl, cyclohexane,norbornane, Z-caproic. In order to facilitate the N-acylation, one tofour glycine residues can be present in the N-terminus of the molecule.

The carboxyl group at the C-terminus of the compound can be protected,for example, by an amide (i.e., the hydroxyl group at the C-terminus isreplaced with —NH ₂, —NHR₂ and —NR₂R₃) or ester (i.e. the hydroxyl groupat the C-terminus is replaced with —OR₂). R₂ and R₃ are independently analiphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or asubstituted aryl group. In addition, taken together with the nitrogenatom, R₂ and R₃ can form a C4 to C8 heterocyclic ring with from about0-2 additional heteroatoms such as nitrogen, oxygen or sulfur. Examplesof suitable heterocyclic rings include piperidinyl, pyrrolidinyl,morpholino, thiomorpholino or piperazinyl. Examples of C-terminalprotecting groups include —NH₂, —NHCH₃, —N(CH₃)₂, —NH(ethyl),—N(ethyl)₂, —N(methyl) (ethyl), —NH(benzyl), —N(C1-C4 alkyl)(benzyl),—NH(phenyl), —N(C1-C4 alkyl) (phenyl), —OCH₃, —O-(ethyl), —O-(n-propyl),—O-(n-butyl), —O-(iso-propyl), —O-(sec-butyl), —O-(t-butyl), —O-benzyland —O-phenyl.

The DJ-1 related peptides of the present invention may be attached(either covalently or non-covalently) to a penetrating agent.

As used herein the phrase “penetrating agent” refers to an agent whichenhances translocation of any of the attached peptide across a cellmembrane.

According to one embodiment, the penetrating agent is a peptide and isattached to the DJ-1 related peptide (either directly or non-directly)via a peptide bond.

Typically, peptide penetrating agents have an amino acid compositioncontaining either a high relative abundance of positively charged aminoacids such as lysine or arginine, or have sequences that contain analternating pattern of polar/charged amino acids and non-polar,hydrophobic amino acids.

Examples of peptide penetrating agents include those set forth in SEQ IDNOs: 21-23. By way of non-limiting example, cell penetrating peptide(CPP) sequences may be used in order to enhance intracellularpenetration. CPPs may included short and long versions of TAT(YGRKKRR—SEQ ID NO: 21 and YGRKKRRQRRR—SEQ ID NO: 22) and PTD (RRQRR—SEQID NO: 23). However, the disclosure is not so limited, and any suitablepenetrating agent may be used, as known by those of skill in the art.

According to a particular embodiment, the peptides of the presentinvention are no longer than 25 amino acids (this includes the DJ-1related peptide together with any additional attached sequence, such asa cell penetrating peptide as described above).

The peptides of the present invention may also comprise non-amino acidmoieties, such as for example, hydrophobic moieties (various linear,branched, cyclic, polycyclic or hetrocyclic hydrocarbons and hydrocarbonderivatives) attached to the peptides; non-peptide penetrating agents;various protecting groups, especially where the compound is linear,which are attached to the compound's terminals to decrease degradation.Chemical (non-amino acid) groups present in the compound may be includedin order to improve various physiological properties such; decreaseddegradation or clearance; decreased repulsion by various cellular pumps,improve immunogenic activities, improve various modes of administration(such as attachment of various sequences which allow penetration throughvarious barriers, through the gut, etc.); increased specificity,increased affinity, decreased toxicity and the like.

Attaching the amino acid sequence component of the peptides of theinvention to other non-amino acid agents may be by covalent linking, bynon-covalent complexion, for example, by complexion to a hydrophobicpolymer, which can be degraded or cleaved producing a compound capableof sustained release; by entrapping the amino acid part of the peptidein liposomes or micelles to produce the final peptide of the invention.The association may be by the entrapment of the amino acid sequencewithin the other component (liposome, micelle) or the impregnation ofthe amino acid sequence within a polymer to produce the final peptide ofthe invention.

The peptides of the invention may be linear or cyclic (cyclization mayimprove stability). Cyclization may take place by any means known in theart. Where the compound is composed predominantly of amino acids,cyclization may be via N- to C-terminal, N-terminal to side chain andN-terminal to backbone, C-terminal to side chain, C-terminal tobackbone, side chain to backbone and side chain to side chain, as wellas backbone to backbone cyclization. Cyclization of the peptide may alsotake place through non-amino acid organic moieties comprised in thepeptide.

The peptides of the present invention can be biochemically synthesizedsuch as by using standard solid phase techniques. These methods includeexclusive solid phase synthesis, partial solid phase synthesis methods,fragment condensation, classical solution synthesis. Solid phasepolypeptide synthesis procedures are well known in the art and furtherdescribed by John Morrow Stewart and Janis Dillaha Young, Solid PhasePolypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Large scale peptide synthesis is described by Andersson Biopolymers2000; 55(3):227-50.

Synthetic peptides can be purified by preparative high performanceliquid chromatography [Creighton T. (1983) Proteins, structures andmolecular principles. WH Freeman and Co. N.Y.] and the composition ofwhich can be confirmed via amino acid sequencing.

Recombinant techniques may also be used to generate the peptides of thepresent invention. To produce a peptide of the present invention usingrecombinant technology, a polynucleotide encoding the peptide of thepresent invention is ligated into a nucleic acid expression vector,which comprises the polynucleotide sequence under the transcriptionalcontrol of a cis-regulatory sequence (e.g., promoter sequence) suitablefor directing constitutive, tissue specific or inducible transcriptionof the polypeptides of the present invention in the host cells.

In addition to being synthesizable in host cells, the peptides of thepresent invention can also be synthesized using in vitro expressionsystems. These methods are well known in the art and the components ofthe system are commercially available.

As mentioned, the peptides of the present invention may be used to treatoxidative-stress related disorders.

As used herein the phrase “oxidative stress” refers to an undesirableimbalance where oxidants outnumber antioxidants. This situation canarise if the rate of ROS production overwhelms existing antioxidantdefenses. In such circumstances, a series of cellular responses canoccur that can lead to an even greater increase in ROS production.Excessive ROS production and its otherwise ineffective regulation can bedetrimental to cells and tissues, inducing cellular damage thatultimately can lead to cell death (apoptosis). Oxidativestress-associated damage also can cause undesirable changes to thestructural and functional integrities of cells that can lead to thepropagation of cells instead of apoptosis. Additionally,oxidatively-damaged cellular macromolecules can trigger immune responsesthat can lead to disease. See generally, D. G. Lindsay et al. (2002)Mol. Aspects of Med. 23:1-38, incorporated herein by reference.

It will be appreciated that oxidative stress may be responsible forinitiating or otherwise causing disease. Alternatively, or additionally,the progression of the disease can be affected by any resultantoxidative stress.

Hence the phrase “oxidative stress related disease” as used herein,refers to a disease or medical condition (including syndromes) whereinthe onset or progression thereof is promoted by oxidative stress. Sinceoxidative stress is believed to be responsible for the pathogenesis ofmany neurological, heart, malignant and age-associated diseases, thepresent invention contemplates all such diseases including for example,atherosclerosis, autoimmune diseases, cancer, cardiovascular disease,cataract, dementia, diabetes and diabetic vasculopathy, andneurodegenerative diseases.

Exemplary neurodegenerative diseases include, but are not limited toParkinson's disease, Multiple Sclerosis, ALS, multi-system atrophy,Alzheimer's disease, stroke, progressive supranuclear palsy,fronto-temporal dementia with parkinsonism linked to chromosome 17 andPick's disease.

The peptides of the present invention may be provided per se or as partof a pharmaceutical composition, where it is mixed with suitablecarriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the DJ-1 related peptidesaccountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular,intracardiac, e.g., into the right or left ventricular cavity, into thecommon coronary artery, intravenous, inrtaperitoneal, intranasal, orintraocular injections.

Conventional approaches for drug delivery to the central nervous system(CNS) include: neurosurgical strategies (e.g., intracerebral injectionor intracerebroventricular infusion); molecular manipulation of theagent (e.g., production of a chimeric fusion protein that comprises atransport peptide that has an affinity for an endothelial cell surfacemolecule in combination with an agent that is itself incapable ofcrossing the BBB) in an attempt to exploit one of the endogenoustransport pathways of the BBB; pharmacological strategies designed toincrease the lipid solubility of an agent (e.g., conjugation ofwater-soluble agents to lipid or cholesterol carriers); and thetransitory disruption of the integrity of the BBB by hyperosmoticdisruption (resulting from the infusion of a mannitol solution into thecarotid artery or the use of a biologically active agent such as anangiotensin peptide). However, each of these strategies has limitations,such as the inherent risks associated with an invasive surgicalprocedure, a size limitation imposed by a limitation inherent in theendogenous transport systems, potentially undesirable biological sideeffects associated with the systemic administration of a chimericmolecule comprised of a carrier motif that could be active outside ofthe CNS, and the possible risk of brain damage within regions of thebrain where the BBB is disrupted, which renders it a suboptimal deliverymethod.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethylcellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (DJ-1 related peptides) effective to prevent,alleviate or ameliorate symptoms of a disorder (e.g., Parkinson'sDisease) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to brain orblood levels of the active ingredient are sufficient to induce orsuppress the biological effect (minimal effective concentration, MEC).The MEC will vary for each preparation, but can be estimated from invitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as is further detailed above.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Materials and Methods

DJ-1 related peptides: Based on bioinformatic data and screening of DJ-1protein several peptide sequences of 20 amino-acid based on DJ-1 may beselected. The peptide sequences that demonstrate neuroprotectiveproperties are designed as peptides #2 and #5 and their sequences are asfollows:

Peptide #2: KGAEEMETVIPVDVMRRAGI SEQ ID NO: 1 Peptide #5:EGPYDVVVLPGGNLGAQNLS SEQ ID NO: 2

Shorter peptide derivatives of Peptide #2 may be synthesized as follows:

KGAEEMETVIPVD; (Pep2a; SEQ ID NO: 3 TVIPVDVMRRAGI; (Pep2b; SEQ ID NO: 4)EMETVIPVDVMRR; (Pep2c; SEQ ID NO: 5) KGAEEMETVIPVDVM; (SEQ ID NO: 6)METVIPVDVMRRAGI; (SEQ ID NO: 7) VDVMRRAGI; (Pep II; SEQ ID NO: 8)KGAEEMETVIPV; (Pep short2a; SEQ ID NO: 9) GAEEME; (Pep I; SEQ ID NO: 10)DVMRRAGI; (Pep short II; SEQ ID NO: 11) TVIPV; (Pep III; SEQ ID NO: 37)VIP. (Pep IV; SEQ ID NO: 12)

Shorter peptide derivatives of Peptide #5 may be synthesized as follows:

DVVVLPGG; (Pep V; SEQ ID NO: 13) EGPYDVVVLPGGN; (SEQ ID NO: 14)VLPGGNLGAQNLS; (SEQ ID NO: 15) DVVVLPGGNLGAQ; (SEQ ID NO: 16)EGPYDVVVLPGGNLG; (SEQ ID NO: 17) VVVLPGGNLGAQNLS; (SEQ ID NO: 18)EGPYDVVVL; (SEQ ID NO: 19) and GNLGAQNLS. (SEQ ID NO: 20)

The peptides are introduced into the cells using PULSin method, usingthe manufacturer's instructions.

The peptides are then attached to cell penetrating peptide (CPP)sequences in order enhance intracellular penetration. CPPs used includedshort and long versions of TAT (YGRKKRR—SEQ ID NO: 21 andYGRKKRRQRRR—SEQ ID NO: 22) and PTD (RRQRR—SEQ ID NO: 23).

Peptide derivatives of peptide #2 (attached to cell penetratingpeptides) are as follows:

SEQ ID NO: 24 Pep 2 TAT a: YGRKKRRKGAEEMETVIPVD SEQ ID NO: 25Pep 2 TAT b: YGRKKRRTVIPVDVMRRAGI SEQ ID NO: 26 Pep 2 TAT c:YGRKKRREMETVIPVDVMRR SEQ ID NO: 27 Pep 2 PTD-5 a: RRQRRKGAEEMETVIPVDVMSEQ ID NO: 28 Pep 2 PTD-5 b: RRQRRMETVIPVDVMRRAGI SEQ ID NO: 29Pep 2 nuc TAT: YGRKKRRQRRRVDVMRRAGI

Peptide derivatives of peptide #5 (attached to cell penetratingpeptides):

SEQ ID NO: 30 Pep5 TAT a: YGRKKRREGPYDVVVLPGGN SEQ ID NO: 31 Pep5 TAT b:YGRKKRRVLPGGNLGAQNLS SEQ ID NO: 32 Pep5 TAT c: YGRKKRRDVVVLPGGNLGAQSEQ ID NO: 33 Pep 5 PTD-5 a: RRQRREGPYDVVVLPGGNLG SEQ ID NO: 34Pep 5 PTD-5 b: RRQRRVVVLPGGNLGAQNLS SEQ ID NO: 35 Pep 5 nuc TAT a:YGRKKRRQRRREGPYDVVVL SEQ ID NO: 36 Pep 5 nuc TAT b: YGRKKRRQRRRGNLGAQNLS

Cells: Human neuroblastoma SH—SY5Y cells are obtained from the Americantissue Type Culture Collection (ATCC, Rockville, USA). Cells are grownunder sterile conditions as monolayer in DMEM medium supplemented with10% heat-inactivated fetal calf serum (FCS), gentamicin (50 mg/ml), andglutamine (5 mM) in a 5% CO₂ humidified atmosphere at 37° C. The mediumis routinely changed every 4 days, and cells are passaged every 8 days.All experiments are performed on cells which are near confluence.

The immortalized human HaCaT keratinocyte cells are used to test DJ-1related peptides ability to protect against UV radiation induced injury.Cells are maintained in minimal essential medium containing 0.075 mMCa²⁺ (MEM-75) and 10% FCS in 5 cm Petri-dishes and subcultured every 3-4days.

Primary cultures and neural stem cells: Neural progenitor/stem cells areisolated from murine cortex of postnatal c57/bl mice and cultured asneurospheres. Neural and astroglial differentiation are induced.

Susceptibility of differentiated and undifferentiated neuralprogenitor/stem cells to oxidative and toxic insults are determined withand without prior pretreatment with DJ-1 related peptides.

Murine neuronal and astrocytes primary cultures derived from corticaltissues of post natal c57/bl mice are prepared and used in order toexamine protective potential of DJ-1 related peptides.

Stable cellular transfections: The coding region of human wild-type orA53T mutant alpha-synuclein cDNA is subcloned into pcDNA3.1-plasmid (BDBiosciences, Clontech). In order to achieve overexpression of A53Tmutant alpha-synuclein, SH—SY5Y neuroblastoma cells are stablytransfected with the plasmid containing A53T mutant alpha-synuclein.Transfections are performed using the lipofectamine 2000 reagent(Invitrogen, Carlsbad, Calif.). Selection of transfected cells isperformed by treating the cells with lethal doses of geniticin (G-418).Naive neuroblastoma cells as well as cells stably transfected with theempty vector are used as controls.

Stable transfections are verified by measuring alpha-synuclein mRNA andprotein levels using real-time PCR and Western blotting.

Treatments: Neuroblastoma cells are contacted with hydrogen peroxide(H₂O₂) (0-1 mM; Sigma, Chemicals Co., St. Louis, Mo., USA),6-hydroxydopamine (0-100 μM; Sigma, Chemicals Co., St. Louis, Mo., USA),dopamine (0-500 μM; Sigma, Chemicals Co., St. Louis, Mo., USA) and3-(4-morpholinyl)-sydnonimine (SIN-I) (0-5 mM; Sigma), (which is aperoxynitrite free radical donor), in order to produce ROS formation.

Cell toxicity and viability assays: Several methods are used in order todetermine the toxic effects of oxidative insults, neurotoxins anddopamine on cellular metabolic activity, mitochondrial activity and cellviability.

Alamar blue: Cells are seeded in 96-wells plates at the concentration of5000 cells per well and allowed to attach over night. On the followingday, the cells are exposed to increasing doses of dopamine (0-500 uM)for 4 hours in serum free medium. Alamar blue is a non toxic reagentwhich incorporates a redox indicator that changes color in response tometabolic activity. The reduction-induced colour change variesproportionately with cell number and time. A solution of 10% alamar blueis added in serum free medium 4 hours following exposure to increasingdoses of dopamine, for 2 hours. Alamar blue fluorescence is measured byFLUOstar spectrofluorometer at the excitation wavelength of 544 nm andthe emission wavelength of 590 nm. Each experiment is performed intriplicate for each treatment. The experiments are repeated 3 times.

Lactate dehydrogenase (LDH) cytotoxicity: LDH released by damaged cellsinto the cell culture supernatant is determined using LDH cytotoxicitydetection kit (Clontech laboratories, CA, USA), according to themanufacturer's instructions. The amount of LDH activity correlates tothe number of damaged cells in the culture. LDH present in the culturesupernatant participates in a coupled reaction converting a yellowtetrazolium salt into a red formazan product. The percentage of deadcells is calculated by the following formula of the absorbance values:

(triplicate absorbance−low control)/(maximum absorption−lowcontrol)×100.

Maximum absorption is obtained by treating the cells with 1% TritonX-100. The amount of enzyme activity is measured in a microplate readerby absorbance at 490 nm. Each experiment is done in triplicate for eachtreatment. The experiment is repeated 3 times.

Hoechst 33342: In order to investigated changes in nuclear morphology ofapoptotic cells, the cells are labeled with the nuclear stain Hoechst33258 and examined under fluorescent microscopy. Hoechst 33342 is a cellfluorescent permeable dye with an affinity for DNA. Following toxinexposure, the cells are fixed with cold 70% ethanol, and incubated withHoechst 33258 (10 ug/ml). Nuclear morphology is observed under afluorescence microscope (Olympus, bx52, Leeds, Minneapolis, Minn.).Cells that exhibited reduced nuclear size, chromatin condensation,intense fluorescence, and nuclear fragmentation are considered to beapoptotic.

Hoechst 33342 enters cells with intact or damaged membranes and stainsDNA in blue, thereby allowing evaluation of cell number in each well aswell. A FLUOstar spectrofluorometer microplate reader may be used inorder to evaluate the cell number in each well following exposure todifferent doses of various toxins. Excitation is performed at 346 nm andemission wavelength is determined at 460 nm. The experiment is performedin triplicate for each treatment. All experiments are repeated at least3 times.

Western blotting: Over expression of WT and mutant alpha-synuclein isdetermined using Western blot. Cells are washed with PBS, trypsinatedand collected by centrifugation. In order to prepare whole-cell lysate,the cells are re-suspended in a lysis buffer (containing 50 mM Tris-HCl,0.1% SDS, 1% Triton X-100, 1 mM EDTA, 1% sodium-deoxycholate and acocktail of protease inhibitors (Sigma)). Protein concentration aredetermined using a protein assay kit (Pierce). Twenty-five micrograms oftotal protein from each sample is separated by 12% SDS-PAGE gels andtransferred to a nitrocellulose membrane. The membranes are blocked in5% non-fat milk for 1 hour in room temperature and incubated overnightat 4° C. with monoclonal anti-alpha-synuclein antibody (LB509, 1:2000;Zymed Laboratories), followed by horseradish peroxidase conjugatedsecondary antibody (1:10000; Sigma) and developed with the ECL plusdetection system (Amersham Pharmacia Biotech). The membranes are alsoincubated with mouse anti beta-actin antibodies (1:10000, Sigma),followed by horseradish peroxidase conjugated secondary antibody(1:10000; Sigma) and developed with the ECL plus detection system, inorder to normalize the alpha-synuclein expression levels to beta-actinlevels.

Immunocytochemistry for alpha-synuclein: Cells were fixed with 4%paraformaldehyde and permeabilized with 0.5% Triton X-100. Subsequently,the fixed cells are incubated in a blocking solution followed byincubation overnight at 4° C. with monoclonal anti-alpha-synucleinantibody (LB509, 1:2000; Zymed Laboratories), followed by alexa-568attached secondary antibodies. Nuclei are counterstained by DAPI(Sigma). The nuclear morphology is observed under a fluorescencemicroscope (Olympus, bx52, Leeds, Minneapolis, Minn.).

Evaluation of apoptosis rate by immunocytochemistry: Cells are fixedwith 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Thenthe fixed cells are incubated in a blocking solution followed byincubation with Hoechst 33258 (10 ug/ml). The nuclear morphology isobserved under a fluorescence microscope (Olympus, bx52, Leeds,Minneapolis, Minn.). Cells that exhibited reduced nuclear size,chromatin condensation, intense fluorescence, and nuclear fragmentationare considered to be apoptotic.

In vivo 6-hydroxydopamine induced hemiparkinsonian mice model: Malec57/bl mice are used for this in vivo model of PD. The mice received aunilateral intrastriatal injection of 4 μg 6-hydroxydopaminehydrobromide (dissolved in 2 μl of saline containing 0.02% ascorbicacid) using a stereotaxic surgical procedure. Injections are targeted tothe central striatum using the following coordinates: 0.7 mm anterior tobregma, 2.0 mm lateral to bregma, and 3.5 mm deep.

Behavioral effects of intrastriatal 6-hydroxydopamine lesioning areevaluated by accelerating rotarod examination and intrperitonealamphetamine (2.5 mg/Kg)-induced rotational behaviour.

Measurements of catecholamine levels by high performance liquidchromatography (HPLC), and the degree of pathological damage, asevaluated by immunohistochemistry, are performed one month following thelesioning.

Statistical analysis: Statistical analysis is done using the SPSSsoftware. Comparisons between two groups are conducted using a 2-tailedStudent's t test. Statistical analyses among three or more groups areperformed using analysis of variance (ANOVA) followed byleast-significant difference (LSD) post hoc comparison. Results arepresented by mean±standard deviation. Differences among groups areconsidered significant if the probability (p) of error was less than 5%(p<0.05).

Example 1 Analysis of Neuroprotective Activity of the Peptides of thePresent Invention as Analyzed in Cell Lines

Results

Tat2A (SEQ ID NO: 24) is tested to determine its protective effectagainst neurotoxins, using an in vitro cellular platform. The peptidesare delivered into neuroblastoma cells by the PULSin kit (Polyplus) andsubsequently, by attachment to cell penetrating peptides. As illustratedin FIGS. 1A-F, some of the examined peptides showed significantprotective effects against serum deprivation, oxidative and neurotoxicinsults induced by exposure to hydrogen peroxide, 6-hydroxydopamine, anddopamine. These peptides reduced mitochondrial damage (evaluated by MTTassay), reduced metabolic damage (indicated by Alamar blue method),decreased cell toxicity (evaluated by LDH cytotoxicity detection kit,Clontech), and increased cell viability (by Hoechst staining).

DJ-1 mutated peptides did not show the protective effect of theircorresponding non-mutated peptides as illustrated in FIGS. 2A-B whichshows the protective effects of several DJ-1 related peptides (*p<0.05).

FIG. 3 illustrates that DJ-1-related peptides are protective in vitroagainst 6-hydroxydopamine toxicity, whereas FIG. 4A illustrates thatDJ-1-related peptides are protective against serum deprivation.

Various non-conserved DJ-1 peptides were used as controls in order toverify the specific effect of the peptides of the present invention.Exposure of neuroblastoma cells to these peptides did not protect thecells from insults induced by 6-hydroxydopamine or hydrogen peroxidetreatments, as illustrated in FIG. 4B.

Example 2 Analysis of Neuroprotective Activity of the Peptides of thePresent Invention as Analyzed in Primary Murine Cultures

Results

T protective effects of DJ-1 peptides on primary cultures, and ondifferentiated and undifferentiated neural stem cells obtained fromC57/bl mice brains were evaluated. Results show that the selected DJ-1related peptides significantly protected against hydrogen peroxide and6-hydroxydopamine toxicity. Specifically, FIGS. 5A-B illustrateprotection of Tat 2A against oxidative and toxic injuries in primarymixed neuronal and astrocytes cultures from post natal mice brains.Neural progenitor/stem cells were isolated from murine cortex ofpostnatal c57/bl mice and cultured as neuro spheres. Neural andastroglial differentiation were induced.

Susceptibility of differentiated and undifferentiated neuralprogenitor/stem cells to oxidative and toxic insults was determined withand without prior pretreatment with DJ-1 related peptides, asillustrated in FIG. 6.

Example 3 Protection Against Mutant Alpha-Synuclein Toxicity

A53T mutant alpha-synuclein is one of the causes for geneticallyinherited Parkinson's Disease. Neuroblastoma cells overexpressingmutated alpha-synuclein were used as a cellular platform for Parkinson'sDisease. In order to achieve overexpression of A53T mutantalpha-synuclein, SH—SY5Y neuroblastoma cells were stably transfectedwith the plasmid containing A53T mutant alpha-synuclein.

Verification of overexpression of alpha-synuclein was effected throughimmunocytochemical staining for alpha synuclein. FIG. 7A illustratesalpha-synuclein staining of naïve neuroblastoma cells, while FIG. 7Billustrates alpha-synuclein staining of A53T mutant alpha-synucleintransfected cells.

Quantification of alpha-synuclein protein was performed using Westernblotting, as illustrated in FIG. 8.

DJ-1 related peptides were shown to be protective against6-hydroxydopamine toxicity in neuroblastoma cells overexpressing mutantA53T alpha-synuclein (FIG. 9) and against pure oxidative insult inducedby hydrogen peroxide (H₂O₂) exposure (FIG. 10).

Example 4 DJ-1 Related Peptides are Protective Against Dopamine Toxicity

Neuroblastoma SH—SY5Y cells were exposed to increasing doses ofdopamine, with or without prior treatment with DJ-1 related peptides.Vulnerability to dopamine was statistically significantly attenuated byDJ-1 related peptides treatment, as illustrated in FIG. 11.

Example 5 Protection Against Cellular Models of Amyotrophic LateralSclerosis (ALS)

NSC-34 cells were used as a cellular model for ALS. Increasing doses ofhydrogen peroxide and SIN-I, an NO donor, which are implicated in thepathogenesis of ALS were used. DJ-1 related peptides #2 showedprotective effects against H₂O₂ and SIN-I toxicity in this cellularmodel of ALS, as illustrated in FIGS. 12A-B and 13.

Example 6 Ability of DJ-1 Related Peptides to Protect Against UVRadiation

Cultured keratinocytes were used to examine the ability of DJ-1 relatedpeptides to protect against the damaging effects of UV radiation.Several DJ-1 related peptides showed significant protective effectsagainst UV radiation induced cell death, as presented in FIG. 14.

Example 7 DJ-1 Related Peptides are Protective Against in Vivo Models ofParkinson's Disease

Recent studies have shown that proteins attached to tat can penetrateand affect the brain after intravenous and intra-peritonealadministration [Kim et al., 2010; Doeppner et al., 2010]. The presentinventors attached peptide 2a, (of 13 amino acids) to tat and tested itin the well established 6-hydroxydopamine hemiparkinsonian mice model.

Next, the protective abilities of intrastriatal injected DJ-1-relatedpeptides were tested. Two groups of male c57/bl mice received either 2μl of 100 μM of TAT 2a DJ-1-related peptide (SEQ ID NO: 24) dissolved insaline or saline alone, injected stereotactically into the rightstriatum. One hour later, 4 μg of 6-hydroxydopamine was injected usingthe same coordinates. Behavioral studies included amphetamine-inducedrotational behavior and accelerating rotarod examinations.

The effect of the DJ-1 related peptide in the 6-hydroxydopamine modelwas tested twice. A total of ten c57/bl male mice were used for thefirst experiment and 26 were used in the second experiment. In both,significantly reduced amphetamine-induced rotations in the peptidetreated mice were found as compared to controls (p<0.01). This wasconfirmed twice in each of the 2 experiments: first, 2 weeks after the6-hydroxydopamine striatal lesioning and subsequently, after 4 weeks, asillustrated in FIGS. 15A-B.

Accelerating Rotarod examination revealed a significant difference(p=0.02) between TAT 2a peptide (SEQ ID NO: 24)-treated mice andsaline-treated controls. Immunohistochemical staining for tyrosinehydroxylase (TH) revealed that the 6-hydroxydopamine-induced loss of THstaining in the lesioned striatum was reversed by tat 2a DJ-1 relatedpeptide (SEQ ID NO: 24).

Next, the effect of systemic administration of the DJ-1-related peptidewas examined. Intravenous delivery of the peptide was performed as amean to modulate 6-hydroxydopamine hemiparkinsonian mice model. 50 μg ofthe peptide was administered intravenously, 4 hours before6-hydroxydopamine lesioning. Compared to controls, a dramatic decreasein amphetamine-induced rotations in the peptide treated mice at 2-weeksand 4-weeks after the lesioning was found (FIGS. 16A-B).

Example 8

In order to generate an animal model for Parkinson's disease, mice weretreated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). 5 mice(C57/bl) were injected for 5 consecutive days with 20 mg/kg MPTP (i.p.)in the presence and absence of the DJ-1 related peptide (0.4 mMsubcutaneous; SEQ ID NO: 25). 18 days later, the mice were sacrificedand the dopamine level, in the two hemispheres was measured by HPLC.

Results

The results of the experiment are demonstrated in FIG. 17.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

1. An isolated peptide comprising a DJ-1 related peptide attached to acell penetrating agent, wherein said DJ-1-related peptide consists ofthe sequence as set forth in SEQ ID NO:
 3. 2. The peptide of claim 1,wherein said cell penetrating agent is a peptide agent.
 3. The peptideof claim 2, wherein said peptide cell penetrating agent comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:21-23.
 4. The peptide of claim 1, comprising the sequence as set forthin SEQ ID NO:
 24. 5. A pharmaceutical composition comprising theisolated peptide of claim 1 as the active agent and a pharmaceuticallyacceptable carrier.
 6. A method of treating an oxidative stress relateddisorder in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of aDJ-1 related peptide attached to a cell penetrating agent, wherein saidDJ-1-related peptide consists of the sequence as set forth in SEQ ID NO:3, thereby treating the oxidative stress related disorder.
 7. The methodof claim 6, wherein said cell penetrating agent is a peptide agent. 8.The method of claim 7, wherein said peptide cell penetrating agentcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 21-23.
 9. The method of claim 6, comprising the sequence asset forth in SEQ ID NO:
 24. 10. The method of claim 6, wherein saidoxidative stress related disorder is a neurodegenerative disorder. 11.The method of claim 6, wherein said neurodegenerative disease isselected from the group consisting of Parkinson's disease, MultipleSclerosis, ALS, multi-system atrophy, Alzheimer's disease, stroke,progressive supranuclear palsy, fronto-temporal dementia withparkinsonism linked to chromosome 17 and Pick's disease.